Cell-based assays that quantitate the effect of small molecules on cellular behavior are powerful tools in cancer research. The ability to assess small molecules in a physiologically relevant environment is informative, as the assays simultaneously evaluate compound permeability, toxicity, and potency. To date, cell-based assays are largely limited to measure global changes such as phenotype, proliferation, or gene expression. The current challenge is to develop cell-based assays for specific enzyme activities to augment and accelerate drug discovery campaigns. Notably, the most common strategies for measuring enzyme activities, such as radioactivity, absorbance, and fluorescent labels, often cannot be delivered to the appropriate target within the cellular environment and they are susceptible to high rates of false positives. This proposal describes the continued development and validation of a novel label-free methodology that combines cell culture and lysis on customized surface chemistries to allow a mass spectrometry readout for distinct enzyme activities. The approach uses functionalized self-assembled monolayers (SAMs) that present cell adhesion ligands along with substrates for the relevant cellular enzyme targets. Cells are cultured on the monolayers in the presence of small molecules, and then lysed such that enzymes within the lysate have immediate access to convert the immobilized substrate to a product. An advantage of using SAMs on gold is their compatibility with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS), an approach termed ?SAMDI?, that records the activity of specific enzymes within the lysate. This technique, termed Tandem Culture and Lysis (TCAL)-SAMDI, offers a novel, powerful tool for a quantitative readout of virtually any cellular enzyme activity and represents new capabilities for high-throughput and label-free, cell- based screens for drug discovery.